Doris Feineis

Biochemical and pharmacological characterization of 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline: a biologically relevant neurotoxin?

Acute and long-term effects of exposure to reactive compounds as the result of environmental pollution, workplace conditions or dietary intake are suspected to be involved in the etiology of a variety of disorders, including neurodegenerative disorders such as Parkinsons disease. The recognition in 1970s that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxic by-product of illicit meperidine synthesis, elicits parkinsonian symptoms in primates, including man, prompted the search for naturally occurring analogs which might be involved in human disease. It has been suggested that one candidate, 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo), a potent dopaminergic neurotoxin, might be formed endogenously in humans following the administration of the hypnotic chloral hydrate or after the exposure to the industrial solvent trichloroethylene. Such spontaneous formation has, indeed, been recently reported. The biochemical and pharmacological characteristics of TaClo and related compounds are thus reviewed here, and their potential significance for human neurodegenerative disease discussed.

1-Trichloromethyl-1,2,3,4-tetrahydro-β-carboline increases extracellular serotonin and stimulates hydroxyl radical production in rats

1-Trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo), a neurotoxin structurally similar to the dopaminergic neurotoxin MPTP, may be formed in humans treated with chloral hydrate or exposed to trichloroethylene, a widely used industrial solvent. Systemically administered TaClo (0.4 mg/kg, i.p.) induced an immediate and transient release of dopamine (DA) and serotonin (5-HT) measured using microdialysis. However, only 5-HT was increased significantly (area under the curve, AUC, for the 1-2 h-period following TaClo administration: 400% compared with the respective control value; 2-3 h-period: 326%). This was followed by a progressive increase in hydroxyl radical formation reflected by higher extracellular concentrations of the hydroxylate product of salicylic acid, 2,3-dihydroxybenzoic acid (AUC for the 1-2 h period following TaClo administration: 182% compared with the respective control value; 2-3 h period: 190%). In contrast, extracellular glutamate and GABA were increased 2-3 h post-injection by 64 and 51%, respectively. These data suggest that TaClo stimulates the generation of hydroxyl free radicals via an acute release of 5-HT and perhaps DA.

Endogenous alkaloids in man XXVI. Determination of the dopaminergic neurotoxin 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo) in biological samples using gas chromatography with selected ion monitoring

Highly chlorinated beta-carbolines have a potential in vivo relevance to Parkinsons disease. In this paper, a gas chromatographic method for the determination of the neurotoxic 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo), the condensation product of tryptamine and chloral hydrate, is described. The specific and sensitive assay involves purification of the biological samples by solid-phase extraction with C18 cartridges, derivatization with heptafluorobutyric anhydride, and chromatography on a non-polar fused-silica capillary column. Detection of TaClo was achieved by the registration of characteristic mass fragments of the TaClo heptafluorobutyric amide derivative using selected ion monitoring. The method was utilized to detect and quantify TaClo in blood, urine, bile, faeces, and brain tissue of rats treated with this alkaloid-type heterocycle. Four-fold deuterium-labelled TaClo was used as an internal standard.

Highly Halogenated Tetrahydro-β-Carbolines as a New Class of Dopaminergic Neurotoxins

The Pictet-Spengler condensation of 2-arylethylamines with aldehydes or α-keto acids (Callaway et al., 1994) is not only the most efficient pathway for the chemical synthesis of tetrahydro-β-carbolines (TH-βCs), but has also been found to play a role in human organisms, leading to endogenous “mammalian alkaloids” (Bringmann, 1979; Buckholtz, 1980; Airaksinen and Kari, 1981a; Airaksinen and Kari, 198 lb; Collins, 1986a; Rommelspacher and Susilo, 1985; Brossi, 1993). During the last three decades, the biological potential of the simple, formaldehyde- and acetaldehyde-derived THβCs tryptoline (1) and eleagnine (2) (see Fig. 1) has been investigated thoroughly because these compounds and related derivatives were regarded as causative links between alcoholism and opioid mania (Davis and Walsh, 1970; Rommelspacher et al., 1984; Myers, 1989). Furthermore, since the merely synthetic neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) was found to induce parkinsonism in humans, monkeys, and mice (Burns et al., 1983; Singer et al., 1993), special attention has been focused on the potential role of THβCs as inducers of parkinsonism, closely related in structure, yet occurring in Nature. Indeed, for several β-carbolines toxic behavior towards dopaminergic neurons was demonstrated (Collins et al., 1986b; Albores et al., 1990; Sayre et al., 1991).

Determination of 1,3-Thiazolidine-carboxylic Acids in Urine by Reversed-Phase HPLC After Fluorescence Labelling with Dansyl Chloride

Abstract Hyperoxaluria (HOU) - a severe inherited disease - is caused by accumulation of the cytotoxic glyoxylic acid. Spontaneous, not enzyme-mediated formation of 1,3-thiazolidines by condensation of glyoxylic acid with sulphur-containing biogenic amines or amino acids is the key step of our “chemical” concept for the therapy of glyoxylate-induced oxalurias. A sensitive and reliable high-performance liquid chromatographic (HPLC) assay for the determination of these highly polar alkaloid-type heterocvcles and their precursors, L-cysteine, cysteamine, and D(-)-penicillamine, has been developed, based on the pre-chromatographic derivatization of secondary amines with dansyl chloride to form yellow fluorescent compounds. Series of tests, monitoring diastereomeric 5,5-dimethyI-thiazoli-dine-2(R,S)-4(S)-dicarboxylic acids after dansylation in matrix-free solution and in urine, respectively, using an external standard method, are presented. The detection limit for urine samples was determined to be 2–3 nmol/ml.

Cytotoxicity of chloral-derived β-carbolines is not specific towards neuronal nor dopaminergic cells

Summary.β-Carbolines structurally related to the selective dopaminergic neurotoxin 1-methyl-4- phenylpyridinium (MPP+) may contribute to dopaminergic neurodegeneration in Parkinson’s disease. The chloral-derived mammalian alkaloid derivative 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo) is formed endogenously by a Pictet-Spengler condensation from the biogenic amine tryptamine (Ta) and the hypnotic aldehyde chloral (Clo). Here we examine the dopaminergic toxicity of TaClo and related compounds by testing their differential cytotoxicities in dopaminergic SH-SY5Y and non-dopaminergic murine Neuro2A neuroblastoma cell lines as well as in heterologous expression systems of the dopamine transporter (DAT) using both HEK-293 and Neuro2A cells. All TaClo derivatives showed significant cytotoxicity in all cell lines after 72 hours with the following rank order of toxic potency: 1-Tribromomethyl-1,2,3,4-tetrahydro-β-carboline (TaBro) > TaClo > MPP+ > 1,2,3,4-tetrahydro-β-carboline (THβC) > 2[N]-methyl-TaClo > 2[N]-methyl-THβC. In contrast to MPP+, there was no selectivity towards dopaminergic cells or cells ectopically expressing the DAT in vitro. Our results suggest that TaClo and related analogs are strong cytotoxins without selectivity towards dopaminergic cells.

Modification of tyrosine hydroxylase activity by chloral derived β‐carbolines in vitro

β‐Carbolines have been suggested to be involved in the pathogenesis of Parkinsons disease as a result of their structural similarity to the neurotoxin N‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP). The chloral‐derived β‐carboline derivative 1‐trichloromethyl‐1,2,3,4‐tetrahydro‐β‐carboline (TaClo) causes cell loss in neuronal and glial cell cultures and induces a slowly developing neurodegenerative process in rats. In our experiments, effects of TaClo and its derivatives 2‐methyl‐TaClo (2‐Me‐TaClo), and 1‐dichloromethylene‐1,2,3,4‐tetrahydro‐β‐carboline (1‐CCl2‐THβC) on tyrosine hydroxylase (TH) activity were investigated in TH assays using homogenate preparations of the rat nucleus accumbens and recombinant human TH (hTH1). TH activity was determined in vitro by measuring l‐DOPA production with HPLC‐ECD. Using homogenate preparations, TaClo, 2‐Me‐TaClo, and 1‐CCl2‐THβC inhibited TH in concentrations of 0.1 mm, while 1‐CCl2‐THβC in low concentrations enhanced TH activity. When TH was activated by PACAP‐27, TaClo, 2‐Me‐TaClo, or 1‐CCl2‐THβC also inhibited activated enzyme activity in high concentrations. However, in the case of 2‐Me‐TaClo and 1‐CCl2‐THβC a biphasic effect was observed with a marked increase of TH activity in the nanomolar range. In our experiments using recombinant hTH1, TaClo, 2‐Me‐TaClo, or 1‐CCl2‐THβC did not modify enzyme activity. After activation of hTH1 by PKA all the tetrahydro‐β‐carbolines investigated in this study decreased l‐DOPA formation. We suggest that these β‐carbolines modulate dopamine synthesis by interacting with a protein kinase TH‐activating system.

Antiplasmodial Ealapasamines A-C,‘Mixed’ Naphthylisoquinoline Dimers from the Central African Liana Ancistrocladus ealaensis

Three unusual heterodimeric naphthylisoquinoline alkaloids, named ealapasamines A-C (1–3), were isolated from the leaves of the tropical plant Ancistrocladus ealaensis J. Léonard. These ‘mixed’, constitutionally unsymmetric dimers are the first stereochemically fully assigned cross-coupling products of a 5,8′- and a 7,8′-coupled naphthylisoquinoline linked via C-6′ in both naphthalene portions. So far, only two other West and Central Ancistrocladus species were known to produce dimers with a central 6,6″-axis, yet, in contrast to the ealapasamines, usually consisting of two 5,8′-coupled monomers, like e.g., in michellamine B. The new dimers 1–3 contain six elements of chirality, four stereogenic centers and the two outer axes, while the central biaryl axis is configurationally unstable. The elucidation of the complete stereostructures of the ealapasamines was achieved by the interplay of spectroscopic methods including HRESIMS, 1D and 2D NMR (in particular ROESY measurements), in combination with chemical (oxidative degradation) and chiroptical (electronic circular dichroism) investigations. The ealapasamines A-C display high antiplasmodial activities with excellent half-maximum inhibition concentration values in the low nanomolar range.

Gardenifolins A–H, Scalemic Neolignans from Gardenia ternifolia: Chiral Resolution, Configurational Assignment, and Cytotoxic Activities against the HeLa Cancer Cell Line

From the tropical plant Gardenia ternifolia Schumach. and Thonn. (Rubiaceae), eight stereoisomeric 2,3-dihydrobenzo[b]furan neolignans, named gardenifolins A-H (1a-d and 2a-d), were isolated and fully structurally characterized. Reversed-phase chromatography of a stem bark extract afforded two peaks, viz. mixtures I and II, each one consisting of two diastereomers and their respective enantiomers. They were resolved and stereochemically analyzed by HPLC on a chiral phase coupled to electronic circular dichroism (ECD) spectroscopy, giving single ECD spectra of all eight stereoisomers. The double-bond geometries (E or Z) of the gardenifolins A-H and their relative configurations (cis or trans) at the stereogenic centers C-7 and C-8 in the dihydrofuran ring system were assigned by 1D and 2D NMR methods, in particular, using NOE difference experiments, whereas the absolute configurations of the isolated enantiomers were established by ECD spectroscopy by applying the reversed helicity rule. The individual pure gardenifolin isomers A-H showed the most different cytotoxic effects against the human cancer HeLa cell line, with 1d and 2a displaying the highest activities, with IC50 values of 21.0 and 32.5 μM, respectively. Morphological experiments indicated that gardenifolin D (1d) induces apoptosis of HeLa cells at 25 μM.

Toxicity and Metabolism of the Chloral-Derived Mammalian Alkaloid 1-Trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo) in PC12 Cells

Chloral-derived β-carbolines, which are structurally similar to the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 5), are discussed to contribute to neuronal cell death in idiopathic Parkinson’s disease. The cytotoxicity of 1-trichloromethyl- 1,2,3,4-tetrahydro-β-carboline (TaClo, 4) to neuronal-like clonal pheochromocytoma PC12 cells was examined by the determination of lactate dehydrogenase (LDH) release. After incubation for 48 h, 4 showed a strong dose-dependent cytotoxic activity towards PC12 cells with an ED50 value of 230 μᴍ. In PC12 cells reductive dehalogenation of 4 was observed giving rise to the formation of 1-dichloromethyl-1,2,3,4-tetrahydro-β-carboline (6) as a main TaClo metabolite exhibiting a cytotoxic potential comparable to that of TaClo. An X-ray structure analysis, performed for the trifluoroacetyl derivative of 6, revealed the N-substituent of such a highly chlorinated agent to be dramatically pushed out of the β-carboline ring ‘plane’ due to the high steric demand of the huge dichloromethyl group at C(1).